Translation of the hepatitis C virus (HCV) genome is mediated by a approximately 350nt.-long internal ribosomal entry site (IRES). Ribosomal subunits (40S) form stable binary complexes with the HCV IRES. The complex structure of this RNA determines the correct positioning of the initiation codon in the ribosomal 'P' site in these complexes. Addition of Met-tRNA, elF2 and GTP to binary complexes results in formation of 48S preinitiation complexes. The aim of this proposal is to characterize how the IRES and components of the cellular translation apparatus interact during initiation, in order to elucidate the unique mechanism of HCV initiation at the molecular level and to identify potential targets in this IRES for chemotherapeutic agents that will inhibit its function. UV cross-linking, chemical footprinting and mutational analyses will be used to identify (i) determinants in the IRES of its interaction with the 40S subunit and (ii) ribosomal proteins and segments of ribosomal RNA that constitute IRES-binding sites on this subunit. Binding determinants will then be matched with binding sites. The same approaches will be used to analyse the conformational rearrangement of the IRES that occurs when eIF2, initiator tRNA and GTP bind to the binary ribosomal complex to form a 48S preinitiation complex. eIF3 (which binds specifically to the IRES) is not required at this stage but is essential for subsequent joining of the 60S subunit to form an active 80S ribosomal complex. Determinants of eIF3's interaction with the IRES will be identified and their importance for eIF3's activity in IRES-mediated initiation will then be assessed in initiation reactions reconstituted in vitro from purified components.